A study on the IFN-?, IL-10 and IL-4 responses obtained by purifying the recombinant NS3 protein of Hepatitis C Virus

Hepatitis-C virus (HCV) is a widespread agent in the world giving rise to public health problems. It is estimated that almost 180 million persons in the world are infected by HCV and it is noteworthy that these are much more eligible to develop first chirrosis and then hepatocellular carcinoma (HCC) as compared to uninfected population by this virus. Positive sense HCV RNA translated as a single large poliprotein (3011 amino acid size) which constitutes 11 different proteins. Non-structural protein 3 (NS3), formed by cutting and processing of this poliprotein by various proteases is one of the largest protein of 631aa, having the serine-protease activity and NTPase/helicase function. Despite the fact that there are numerous studies on the participation of HCV NS3 protein in the inhibition of intercellular pathways and its role on the evading immune system, current data on the development of cytokine response against NS3 is insufficient.In this study, purification of a local HCV-based NS3 protein and use of this protein in the studies of cytokine-response against the NS3, is aimed for the first time in Turkey. For his purpose, HCV RNA is used as a template, isolated from a patient carrying HCV genotype-1, in the Dokuz Eylul University Hospital Central Laboratories.The region in HCV-RNA which coding the NS3 is amplified by RT-PCR and inserted by ligation reaction into expression vector and expressed in the full length HCV NS3 Escherichia coli protein M15 cells. pQE30 plasmid is utilized containing 6xHis-Tag as the expression vector. Recombinant NS3 is purified by agarose- nickel-nitrilotriasetic acid (Ni?NTA) chromatography under denatured conditions.For the the cytokine response studies, whole blood and peripheral blood mononuclear cells of the study group, consisting of 4 men and 3 women with good health, stimulated by recombinant NS3. In the supernatants of the stimulated and non-stimulated cells, IFN?, IL-4, IL-10 cytokine responses are evaluated by utilizing quantitative ELISA method.The ELISA results of cytokine assays after stimulation whith NS3, indicate that significantly high IL-10 response occurs after the stimulation against the recombinant NS3 protein, whereas same response is not present for IFN? and IL-4 especially with whole blood samles.In summary, after scanning Turkish and in international literature, a HCV-genotype-1 based NS3 protein is purified for its first time, and optimization and standardization studies on the responses of cytokine are already in progress.